The 9-1-1 DNA Clamp Is Required for Immunoglobulin Gene Conversion

ABSTRACT Chicken DT40 cells deficient in the 9-1-1 checkpoint clamp exhibit hypersensitivity to a variety of DNA-damaging agents. Although recent work suggests that, in addition to its role in checkpoint activation, this complex may play a role in homologous recombination and translesion synthesis, the cause of this hypersensitivity has not been studied thoroughly. The immunoglobulin locus of DT40 cells allows monitoring of homologous recombination and translesion synthesis initiated by activation-induced deaminase (AID)-dependent abasic sites. We show that both the RAD9 −/− and RAD17 −/− mutants exhibit substantially reduced immunoglobulin gene conversion. However, the level of nontemplated immunoglobulin point mutation increased in these mutants, a finding that is reminiscent of the phenotype resulting from the loss of RAD51 paralogs or Brca2. This suggests that the 9-1-1 complex does not play a central role in translesion synthesis in this context. Despite reduced immunoglobulin gene conversion, the RAD9 −/− and RAD17 −/− cells do not exhibit a prominent defect in double-strand break-induced gene conversion or a sensitivity to camptothecin. This suggests that the roles of Rad9 and Rad17 may be confined to a subset of homologous recombination reactions initiated by replication-stalling lesions rather than those associated with double-strand break repair.

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NHEJ-deficient DT40 cells have increased levels of immunoglobulin gene conversion: evidence for a double strand break intermediate

Nucleic Acids Research ◽

10.1093/nar/gkl830 ◽

2006 ◽

Vol 34 (21) ◽

pp. 6345-6351 ◽

Cited By ~ 16

Author(s):

Ephraim S. Tang ◽

Alberto Martin

Keyword(s):

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

Immunoglobulin Gene ◽

Dt40 Cells

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Genetic Requirements for RAD51- andRAD54-Independent Break-Induced Replication Repair of a Chromosomal Double-Strand Break

Molecular and Cellular Biology ◽

10.1128/mcb.21.6.2048-2056.2001 ◽

2001 ◽

Vol 21 (6) ◽

pp. 2048-2056 ◽

Cited By ~ 104

Author(s):

Laurence Signon ◽

Anna Malkova ◽

Maria L. Naylor ◽

Hannah Klein ◽

James E. Haber

Keyword(s):

Saccharomyces Cerevisiae ◽

Homologous Recombination ◽

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

Telomere Maintenance ◽

Diploid Cells ◽

Break Induced Replication ◽

In Cells

ABSTRACT Broken chromosomes can be repaired by several homologous recombination mechanisms, including gene conversion and break-induced replication (BIR). In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) is normally repaired by gene conversion. Previously, we have shown that in the absence ofRAD52, repair is nearly absent and diploid cells lose the broken chromosome; however, in cells lacking RAD51, gene conversion is absent but cells can repair the DSB by BIR. We now report that gene conversion is also abolished when RAD54, RAD55, and RAD57 are deleted but BIR occurs, as withrad51Δ cells. DSB-induced gene conversion is not significantly affected when RAD50, RAD59, TID1(RDH54), SRS2, or SGS1 is deleted. Various double mutations largely eliminate both gene conversion and BIR, including rad51Δ rad50Δ, rad51Δ rad59Δ, andrad54Δ tid1Δ. These results demonstrate that there is aRAD51- and RAD54-independent BIR pathway that requires RAD59, TID1, RAD50, and presumablyMRE11 and XRS2. The similar genetic requirements for BIR and telomere maintenance in the absence of telomerase also suggest that these two processes proceed by similar mechanisms.

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Role for RAD18 in Homologous Recombination in DT40 Cells

Molecular and Cellular Biology ◽

10.1128/mcb.01291-06 ◽

2006 ◽

Vol 26 (21) ◽

pp. 8032-8041 ◽

Cited By ~ 36

Author(s):

Dávid Szüts ◽

Laura J. Simpson ◽

Sarah Kabani ◽

Mitsuyoshi Yamazoe ◽

Julian E. Sale

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Gene Conversion ◽

Early Stage ◽

Current Evidence ◽

Recombination Reaction ◽

Immunoglobulin Gene ◽

Postreplication Repair ◽

Strand Breaks ◽

Dt40 Cells

ABSTRACT RAD18 is an E3 ubiquitin ligase that catalyzes the monoubiquitination of PCNA, a modification central to DNA damage bypass and postreplication repair in both yeast and vertebrates. Although current evidence suggests that homologous recombination provides an essential backup in vertebrate rad18 mutants, we show that in chicken DT40 cells this is not the case and that RAD18 plays a role in the recombination reaction itself. Gene conversion tracts in the immunoglobulin locus of rad18 cells are shorter and are associated with an increased frequency of deletions and duplications. rad18 cells also exhibit reduced efficiency of gene conversion induced by targeted double-strand breaks in a reporter construct. Blocking an early stage of the recombination reaction by disruption of XRCC3 not only suppresses immunoglobulin gene conversion but also prevents the aberrant immunoglobulin gene rearrangements associated with RAD18 deficiency, reverses the elevated sister chromatid exchange of the rad18 mutant, and reduces its sensitivity to DNA damage. Together, these data suggest that homologous recombination is toxic in the absence of RAD18 and show that, in addition to its established role in postreplication repair, RAD18 is also required for the orderly completion of gene conversion.

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Mechanisms for gene conversion and homologous recombination: The double-strand break repair model and the successive half crossing-over model

Advances in Biophysics ◽

10.1016/0065-227x(92)90023-k ◽

1992 ◽

Vol 28 ◽

pp. 81-133 ◽

Cited By ~ 32

Author(s):

I Kobayashi

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Crossing Over ◽

Break Repair

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Mitotic gene conversion tracts associated with repair of a defined double-strand break in Saccharomyces cerevisiae

10.1101/132167 ◽

2017 ◽

Author(s):

Yee Fang Hum ◽

Sue Jinks-Robertson

Keyword(s):

Saccharomyces Cerevisiae ◽

Homologous Recombination ◽

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

The Other ◽

Homologous Chromosomes ◽

Molecular Features ◽

Single Side ◽

Mitotic Gene Conversion

AbstractMitotic recombination between homologous chromosomes can lead to loss-of-heterozygosity (LOH), which is an important contributor to human disease. In the current study, a defined double-strand break (DSB) on chromosome IV was used to initiate LOH in a yeast strain with sequence-diverged chromosomes. Associated gene conversion tracts, which reflect the repair of mismatches formed when diverged chromosomes exchange single strands, were mapped using microarrays. LOH events reflected two broken chromosomes, one of which was repaired as a crossover and the other as a noncrossover. Gene conversion tracts associated with individual crossover and noncrossover events were similar in size and position, with half of the tracts unexpectedly mapping to only a single side of the initiating break. Although the molecular features of DSB-initiated events generally agree with those predicted by current models of homologous recombination, there were unexpected complexities in associated gene conversion tracts.

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Fanconi Anemia Protein FANCD2 Promotes Immunoglobulin Gene Conversion and DNA Repair through a Mechanism Related to Homologous Recombination

Molecular and Cellular Biology ◽

10.1128/mcb.25.1.34-43.2005 ◽

2005 ◽

Vol 25 (1) ◽

pp. 34-43 ◽

Cited By ~ 98

Author(s):

Kazuhiko Yamamoto ◽

Seiki Hirano ◽

Masamichi Ishiai ◽

Kenichi Morishima ◽

Hiroyuki Kitao ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Homologous Recombination ◽

Gene Conversion ◽

Fanconi Anemia ◽

Strand Break ◽

Immunoglobulin Gene ◽

Dna Double Strand Break ◽

Mutant Cells ◽

Light Chain Locus

ABSTRACT Recent studies show overlap between Fanconi anemia (FA) proteins and those involved in DNA repair mediated by homologous recombination (HR). However, the mechanism by which FA proteins affect HR is unclear. FA proteins (FancA/C/E/F/G/L) form a multiprotein complex, which is responsible for DNA damage-induced FancD2 monoubiquitination, a key event for cellular resistance to DNA damage. Here, we show that FANCD2-disrupted DT40 chicken B-cell line is defective in HR-mediated DNA double-strand break (DSB) repair, as well as gene conversion at the immunoglobulin light-chain locus, an event also mediated by HR. Gene conversions occurring in mutant cells were associated with decreased nontemplated mutations. In contrast to these defects, we also found increased spontaneous sister chromatid exchange (SCE) and intact Rad51 foci formation after DNA damage. Thus, we propose that FancD2 promotes a subpathway of HR that normally mediates gene conversion by a mechanism that avoids crossing over and hence SCEs.

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